BINDING OF THE BRUCELLA-ABORTUS LIPOPOLYSACCHARIDE O-CHAIN FRAGMENT TO A MONOCLONAL-ANTIBODY - QUANTITATIVE-ANALYSIS BY FLUORESCENCE QUENCHING AND POLARIZATION

An antigenic O-chain polysaccharide fragment derived from Brucella abo rtus lipopolysaccharide was labeled with 14.8 +/- 1.8 (n = 5) and 52.3 +/- 2.4 (n = 3) mu mol of fluorescein/g of polysaccharide (designated FL(1) and FL(2), respectively) for use in investigating the binding o f O-chain to a specific murine antibody YsT9 under equilibrium conditi ons. Upon binding to YsT9, the fluorescence of FL(1) and FL(2) was que nched 45-57% with no shift in the excitation and emission spectra, and polarization of fluorescence increased by 300-335%. With fluorescence quenching and polarization as sensitive signals for antibody-bound la beled O-chains, the equilibrium constants for binding of FL(1), FL(2), and unlabeled O-chain to YsT9 were determined to be within a similar order (1.5 x 10(7) to 2.0 x 10(7) M(-1)) using a nonlinear curve fitti ng approach rather than Scatchard analysis. These results indicated th at covalent attachment of fluorescein groups to the O-chain did not in fluence the recognition of the YsT9-defined epitope by the antibody. T he reversibility of the O-chain-antibody reaction was also demonstrate d by showing a rapid depolarization of the labeled O-chain-antibody co mplex in the presence of unlabeled O-chain, suggesting that this displ acement experiment could be exploited to quantify the Brucella polysac charide antigen. The study described here provides a useful model for characterization of the complex formation between a carbohydrate-bindi ng protein and a carbohydrate ligand and also for the design of a homo geneous assay system to quantitate antigens or antibodies of clinical interest.